1. Field of the Invention
The present invention relates to cyclodextrin glycosyl transferases (CGTases) EC 2.4.1.19 for producing xcex3-cyclodextrin, to processes for preparing xcex3-cyclodextrin glycosyl transferases, and to their use.
2. The Prior Art
As a rule, cyclodextrins are prepared from starch or starch-like substrates. In these preparations, CGTases are used to convert starch enzymically into cyclodextrin (CD). For thermodynamic reasons, the starch is mainly converted into xcex2-CD, independently of the CGTase used for the reaction, if the reaction is carried out until the thermodynamic equilibrium is reached (maximum CD yield). However, in the initial phase, at the beginning of the starch conversion reaction, the enzymes which are used for the conversion differ in the composition of the primary product mixture. xcex1- xcex2- or xcex3-CGTases are differentiated depending upon the product, xcex1, xcex2-, or xcex3-CD, which is chiefly formed by the enzyme in this initial phase.
These enzymes, which are suitable, and have also already been used, for the industrial production of CD, have hitherto only been detected in bacteria. xcex1-CGTases have hitherto only been identified in Bacillus macerans, Bacillus stearothermophilus and Klebsiella oxytoca. xcex2-CGTases have been detected, for example, in Bacillus circulans, Bacillus megaterium, Bacillus ohbensis, Micrococcus sp. and alkalophilic Bacillae which have not been precisely classified taxonomically, such as Bacillus sp. 38-2, 17-1, 1011 or 1-1. Naturally occurring enzymes having an initially high xcex3-CD-forming activity have been reported in Bacillus subtilis 313, Bacillus sp. A1-6 and Bacillus sp. 290-3.
Since the CGTases which are used in the industrial preparation of cyclodextrins always yield mixtures of several cyclodextrins when converting starch into cyclodextrins, various processes have been developed for isolating pure cyclodextrins (xcex1, xcex2 or xcex3). These are described below:
Defined CDs can be separated out chromatographically from the product mixtures, e.g. on the basis of differences in their molecular weights (described, for example, in U.S. Pat. No. 4,808,232).
As a rule, when starch is converted enzymically into cyclodextrins, complexing agents are added which only react with one defined CD and with this CD form an insoluble complex, for example, which can then be separated out from the reaction mixture by physical means. Subsequently, the complex is resolved and the homogeneous CD is isolated (described, for example, in EP 0291067).
When a xcex3-CGTase is used, the product composition can be displaced in the xcex3-CD direction by adding an organic solvent, such as ethanol, to the reaction mixture (J. Ferrm. Bioeng. (1990) 70 (3), pp. 150-154).
In each of the processes, those CGTases are optimally used which possess an initial product formation preference which is as high as possible for the CD which is to be prepared in pure form.
The specificity of the previously known xcex1- and xcex2-CGTases is adequate for industrial production of the corresponding cyclodextrins. By contrast, none of the known, naturally occurring xcex3-CGTases possesses a product specificity which permits a comparable industrial production of xcex3-CD.
In order to prepare xcex3-CD, therefore, it was proposed, in CA 115:157165, that xcex1- and/or xcex2-Cyclodextrins be converted enzymically into xcex3-CD by adding the xcex3-CD-specific complexing agent glycosyl glycyrrhizin, maltose and a CGTase.
Another option for preparing xcex3-CD consists in increasing the xcex3-CD specificity of xcex2-CGTase, by means of exchanging defined amino acid residues, to such a degree that the mutagenized enzyme produces xcex3-CD to an increased. extent and can consequently be used for preparing xcex3-CD on an industrial scale. Appropriate mutations are known and described, for example, in DE 43 24 650 A1 (corresponds to U.S. Pat. No. 5,474,917), Biochemistry (1994) 33 (33), pp. 9929-9936, Biochemistry (1995) 34 (10), pp. 3368-3376 and J. Biotech. (1994) 32, pp. 283-288.
Such CGTase derivatives, which have been produced by mutagenizing xcex2-CGTases, possess an increased specificity for xcex3-CD and are consequently, on the basis of their product spectra, suited, in principle, for the industrial preparation of xcex3-CD. However, a disadvantage is that the specific activities of the starting enzymes which are used for the mutagenesis are reduced by introducing the relevant mutations. In dependence on the amino acid residues which are introduced, mutated enzymes having an increased specificity for xcex3-CD only possess between 25% and 50% of the CD-forming activity of the starting enzyme (Biochemistry (1994) 33 (33), pp. 9929-9936, Biochemistry (1995) 34 (10), pp. 3368-3376).
It is an object of the present invention to provide cyclodextrin glycosyl transferases (CGTases) which, when converting starch or starch-like substrates into CD, produce xcex3-CD to an increased extent and which still exhibit at least 60% of the specific total CGTase activity of the starting CGTase which was used for preparing the enzyme concerned.
An additional object of the present invention is to provide processes for preparing the said CGTases.
A further object of the present invention is to provide a process for producing xcex3-CD.
The first-mentioned object is achieved by CGTases whose amino acid sequence. differs from the amino acid sequence of wild-type CGTases by the deletion of from 3 to 8 amino acids in the region from amino acid position 155 up to and including amino acid position 195, where position 1 of the protein sequence is the beginning of the signal peptide of the CGTase and the deletion increases the xcex3-CGTase activity of the protein.
Within the meaning of the invention, increases in the xcex3-CGTase activity is understood to mean that the quotient       quantity    ⁢          xe2x80x83        ⁢    of    ⁢          xe2x80x83        ⁢    γ    ⁢          -        ⁢    CD    ⁢          xe2x80x83        ⁢    formed        (                  quantity        ⁢                  xe2x80x83                ⁢        of        ⁢                  xe2x80x83                ⁢        α        ⁢                  -                ⁢        CD        ⁢                  xe2x80x83                ⁢        formed            +              quantity        ⁢                  xe2x80x83                ⁢        of        ⁢                  xe2x80x83                ⁢        β        ⁢                  -                ⁢        CD        ⁢                  xe2x80x83                ⁢        formed              )  
becomes greater in the product mixture which arises when starch or starch-like substrates are reacted with CGTases.
Preferably, the amino acid sequences of CGTases according to the invention differ from the amino acid sequences of known CGTases by between three and eight amino acid residues being deleted in the region between amino acid position 155 and amino acid position 195 of their protein sequence, where position 1 of the protein sequence is the beginning of the signal peptide of the CGTase and the deletion increases the xcex3-CGTase activity of the protein.
Particularly preferably, the amino acid sequences of CGTases according to the invention differ from the amino acid sequences of known CGTases by five amino acid residues being deleted in the region between amino acid position 155 and amino acid position 195 of their protein sequence, where position 1 of the protein sequence is the beginning of the signal peptide of the CGTase and the deletion increases the xcex3-CGTase activity of the protein.
It also applies for each of the other amino acid positions mentioned in the application that position 1 of the protein sequence is the beginning of the signal peptide of the CGTase.
In addition, CGTases are in particular preferred whose amino acid sequences differ from the amino acid sequences of the CGTases specified in Table 1 and FIG. 1 at least by the deletion of the amino acid residues which are in each case printed in bold, with the remaining amino acid sequence of the respective CGTase according to the invention being homologous to the amino acid sequence of the CGTase specified in Table 1 and FIG. 1 to the extent that the sequence exhibits CGTase activity without the deletion according to the invention.
Examples of CGTases according to the invention are CGTases which are obtained from the CGTases listed in Table 1 and FIG. 1, or from other CGTases, by deleting individual amino acid residues in the region between the amino acid residues 155 and 195. CGTases are preferred in which from four to eight residues have been deleted from the said region. CGTases are particularly preferred in which the six amino acids marked by bold type in Table 1 and FIG. 1 have been deleted from the said region.
Further examples of CGTases according to the invention are enzymes from which the amino acids which are homologous to the amino acids specified in Table 1 and FIG. 1 have been deleted, with these enzymes exhibiting CGTase activity without the deletion according to the invention.
Further, examples of CGTases according to the invention are enzymes in which the amino acid residues which are in each case printed in bold in FIG. 1 have been deleted from the region between amino acid position 155 and amino acid position 195, with the remaining amino acid sequence of the CGTases according to the invention being homologous to the amino acid sequence of the CGTase from the microorganism which is in each case specified in FIG. 1 and Table 1 to the extent that the enzyme whose sequence does not contain the deletion according to the invention exhibits CGTase activity.
The between three and eight amino acid residues of (SEQ ID NO: 6-13) in Table 1 are these deleted portions found in (SEQ ID NO:3). The six amino acid residues of (SEQ ID NO:14) is that deleted portion found in (SEQ ID NO:2). The six amino acid residues of (SEQ ID NO:15) is that deleted portion found in (SEQ ID NO:3). The six amino acid residues of (SEQ ID NO:16) is that deleted portion found in (SEQ ID NO:4). The three and five amino acid residues of (SEQ ID NO:17-19) in Table 1 are these deleted portions found in (SEQ ID NO:5).
The CGTase protein of the invention has an amino acid sequence which differs from the amino acid sequences of the CGTases specified in FIG. 1 and Table 1 at least by the deletion of the amino acid residues which are selected from the group consisting of:
(SEQ ID NO:6) deleted from (SEQ ID NO:1);
(SEQ ID NO:7) deleted from (SEQ ID NO:1);
(SEQ ID NO:8) deleted from (SEQ ID NO:1);
(SEQ ID NO:9) deleted from (SEQ ID NO:1);
(SEQ ID NO:10) deleted from (SEQ ID NO:1);
(SEQ ID NO:11) deleted from (SEQ ID NO:1);
(SEQ ID NO:12) deleted from (SEQ ID NO:1);
(SEQ ID NO:13) deleted from (SEQ ID NO:1);
(SEQ ID NO:14) deleted from (SEQ ID NO:2);
(SEQ ID NO:15) deleted from (SEQ ID NO:3).;
(SEQ ID NO:16) deleted from (SEQ ID NO:4);
(SEQ ID NO:17) deleted from (SEQ ID NO:5);
(SEQ ID NO:18) deleted from (SEQ ID NO:5) and
(SEQ ID NO:19) deleted from (SEQ ID NO:5).
The remaining amino acid sequence of the CGTase according to the invention is homologous to the amino acid sequence of the CGTase from the microorganism which is in each case specified in FIG. 1 to the extent that the sequence exhibits CGTase activity without the deletion according to the invention.
Incorporated herewith are five references which publish the complete sequence of the five GCTases compared in FIG. 1 of the application. These five references are as follows: xe2x80x9cCloning and Nucleotide Sequence of a Thermostable Cyclodextrin Glycosyltransferase Gene from Thermoanaerobacter sp. ATCC 53627 and Its Expression in Escherichia colixe2x80x9d, S. T. Jorgensen et al, Biotechnology Letters, Vol. 19, No. 10, October 1997, pages 1027-1031; xe2x80x9cCloning and Sequencing of a Cyclodextrin Glucanotransferase Gene from Bacillus ohbensis and Its Expression in Escherichia coli*xe2x80x9d, Sin et al., Applied Microbiology and Biotechnology (1991) 35:600-605; xe2x80x9cHighly Homologous Cyclodextrin Glycosyltransferases from Bacillus circulans Strain 8 and a Strain of Bacillus licheniformisxe2x80x9d, Bender, Applied Microbiology and Biotechnology (1990) 34:229-230; xe2x80x9cMolecular Cloning, DNA Nucleotide Sequencing, and Expression in Bacillus subtilis Cells of the Bacillus macerans Cyclodextrin Glucanotransferase Genexe2x80x9d, Takano, et al., Journal of Bacteriology, vol. 166, No. 3, June 1986, pages 1118-1122; and xe2x80x9cCloning and Nucleotide Sequence of a Cyclodextrin it Glycosyltransferase Gene from the Alkalophilic Bacillus 1-1xe2x80x9d, O. Huber and J. Szejtli (eds.), Proceedings of the Fourth International Symposium on Cyclodextrins, 71-76, (copyright)1988 by Kluwer Academic Publishers. Please note that the published sequence of B. macerans (=Paenibacillus macerans) includes several errors and that the correct sequence must be taken from the printout of the Swissprot-Database, also enclosed. These sequences and the complete publications are herewith incorporated by reference into the present application. The word xe2x80x9cregionxe2x80x9d can also be substituted by xe2x80x9camino acid sequence(s)xe2x80x9d or xe2x80x9cpeptidexe2x80x9d or xe2x80x9cpart of the CGTasesxe2x80x9d.
To define xe2x80x9chomologousxe2x80x9d, it can be stated that homologous amino acid residues are determined as follows: The amino acid sequence of a respective CGTase has to be compared with the five CGTases as shown in FIG. 1 using the computer program xe2x80x9cpile upxe2x80x9d (Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis.). Using the present standard parameters, the program generates a multiple alignment of the six CGTase sequences. Amino acid residues which are located at the same position in such an alignment are called xe2x80x9chomologousxe2x80x9d.
The wording xe2x80x9cposition 155 and 195xe2x80x9d was chosen to solve the problem resulting from the different absolute position of defined homologous amino acids in different CGTases. According to the teaching of the present application, a region of 8 amino acids is essential for the invention. Deletion within these 8 amino acids results in a change of the product specificity of the CGTase. Just because the absolute position of these 8 amino acids is not the same in all CGTases, the present application defines a sequence region (positions 155 to 195) in which the 8 amino acids essential for the invention can be found. Thus by the wording used in the present application, the region essential for the present application is defined independently from the absolute position in a CGTase.
Unexpectedly, the CGTases according to the invention possess a higher xcex3-CD specificity than that of the starting CGTases which were used for their preparation while, at the same time, the mutated enzyme only exhibits an insignificant reduction in specific total CGTase activity as compared with that of the starting CGTase.
When converting starch or starch-like substrates, the CGTases according to the invention consequently produce CDs in a product distribution in which the quotient of xcex3-CD and the sum of xcex1-CD and xcex2-CD is greater than the quotient of these products which is obtained when starch is converted using the respective unaltered starting CGTase.
The list shown in Table 1 and in FIG. 1 shows, using a few CGTases by way of example, the homologous amino acid sequence region which is generally present in CGTases and the six amino acid residues within this sequence region which are in each case relevant for modifying the product specificity.
The four amino acid sequences shown in FIG. 1 are the same respectively as the four amino acid sequences shown in Table 1.
The number of the first amino acid of each of the amino acid sequences depicted in Table 1 and in FIG. 1 is designated as the position, with the first amino acid of the signal peptide of the particular CGTase sequence having been counted as position 1. The corresponding sequence region can be found in all CGTases using well known standard methods. This can be done, for example, using known algorithms which calculate multiple sequence alignments. An example of a suitable computer algorithm is the xe2x80x9cpileupxe2x80x9d program from the commercially available Wisconsin Sequence Analysis package (Genetic Computer Group, Madison, Wisconsin) sequence analysis program.
By means of mutagenizing the depicted region in CGTases, enzymes according to the invention can be prepared from any CGTases using known standard methods, as explained, by way of example, in the present application. For this purpose, a gene encoding a CGTase is as a rule mutated in such a way that it then encodes a CGTase according to the invention.
The invention consequently also relates to processes for preparing mutated CGTase genes which encode CGTases according to the invention, wherein the DNA sequence of a gene encoding a starting CGTase is mutated, by means of mutagenesis methods which are known per se, such that the amino acid sequence in the region between amino acid positions 155 and 195, which is encoded by the DNA sequence of the mutated gene, differs from the amino acid sequence which is encoded by the DNA of the unmutated gene by the deletion of at least one amino acid residue.
Preferably, in the process according to the invention, the DNA sequence of a gene encoding a starting CGTase is mutated, by means of mutagenesis methods which are known per se, such that the amino acid sequence encoded by the DNA sequence of the mutated gene differs from the amino acid sequence encoded by the DNA of the unmutated gene by the deletion of from four to eight amino acid residues from the region between amino acid positions 155 and 195.
Particularly preferably, in the process according to the invention, the DNA sequence of a gene encoding a starting CGTase is mutated, by means of mutagenesis methods which are known per se, such that the amino acid sequence encoded by the DNA sequence of the mutated gene differs from the amino acid sequence encoded by the DNA of the unmutated gene by the deletion of six amino acid residues from the region between amino acid positions 155 and 195.
The invention furthermore relates to processes for preparing xcex3-CGTases, wherein at least one of the described DNA sequences is expressed in a microorganism.
The genes of all CGTases (starting CGTases) are suitable for preparing the CGTases according to the invention. While starting CGTases can be all naturally occurring CGTases, they can also be CGTases which are obtained by mutagenesis, for example those CGTases in which the product formation ratio has already been altered by another mutation which is not in accordance with the invention (e.g.: as in DE 43 24 650 A1, which corresponds to U.S. Pat. No. 5,474,917). Starting CGTases are preferably those CGTases in which the product formation ratio has already been altered by another mutation which is not in accordance with the invention (e.g.: as described in DE 43 24 650 A1).
The gene encoding a starting CGTase is isolated using known methods and the mutation according to the invention is introduced into the gene of the CGTase by xe2x80x9cin-vivoxe2x80x9d or xe2x80x9cin-vitroxe2x80x9d mutagenesis methods. These methods are likewise well known in the state of the art.
xe2x80x9cIn-vivoxe2x80x9d mutagenesis methods are to be understood as being, in particular, those methods in which microorganisms which chromosomally and/or episomally contain a gene encoding a CGTase are mutagenized in a non-specific manner with a mutagen such as UV light, nitrosoguanidine or ethyl methyl sulfonate. Such a method is described, for example, by Miller J. H. in (1972) Experiments in Molecular Genetics; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y.
Subsequently, known methods, such as sequence analysis in accordance with the chain termination method described by Sanger et al. in PNAS 74 (1977) 5463-5467 are used to identify mutants in which at least one codon of the CGTase gene, encoding an amino acid residue, has been deleted from the region between amino acid residues 155 and 195 of the corresponding CGTase.
Those mutants are preferably selected in accordance with the invention in which from four to eight codons have been deleted from the said region.
Mutants are particularly preferably selected in which six codons have been deleted, with those mutants once again preferably being selected in which the six codons have been deleted which encode the amino acid residues which are printed in bold in FIG. 1 or encode the amino acid residues which are homologous to these residues in other CGTases.
Within the meaning of the invention, xe2x80x9cin-vitroxe2x80x9d mutagenesis methods are to be understood as being those methods in which an isolated CGTase gene, or a fragment of a CGTase gene, is modified, in a manner known per se, such that a gene is produced which encodes a CGTase enzyme in which at least one codon of the CGTase gene, encoding one of the amino acid residues in the region between amino acid residues 155 and 195, has been deleted.
Mutants are preferred which have been modified such that from four to eight codons have been deleted in the said region. Mutants are particularly preferred which have been modified such that six codons have been deleted in the said region. In particular, mutants are particularly preferred which have been modified such that, in the said region, the six codons have been deleted which encode the amino acid residues which are printed in bold in FIG. 1 or encode the amino acid residues which are homologous to these residues in other CGTases.
The invention consequently also relates to DNA sequences which encode xcex3-CGTases according to the invention.
Examples of methods for xe2x80x9cin-vitroxe2x80x9d mutagenesis which are known from the state of the art are specific (BioTechniques (1992) 13 (3), pp. 342-346) or non-specific (Technique (1989) 1 (1), pp. 11-15) mutagenesis methods which use the xe2x80x9cPCRxe2x80x9d technique. Methods are also known in which the mutation is introduced into the target gene in a directed manner using a synthetic oligonucleotide. This can take place either using so-called xe2x80x9csingle-strand methodsxe2x80x9d (Ausubel F. M. et al. (1987) Current Protocols in Molecular Biology, Green Publishing Associates) or using xe2x80x9cdouble-strand methodsxe2x80x9d (Promega 1992-1993 Catalogue, 150) or using other methods as described, for example, in Ann. Rev. Genet. (1985) 19, pp. 423-462.
The main area of application for the CGTase according to the invention is its use for isolating xcex3-CD from starch. The CGTases according to the invention can be employed for this purpose using current preparation methods.
The invention consequently also relates to processes for preparing xcex3-CD by converting starch using a CGTase, wherein at least one CGTase according to the invention is employed as the CGTase.
Current preparation methods for producing xcex3-CD, in which the CGTases according to the invention can be employed in place of the CGTases which are specified in these methods, are described, for example, in:
Journal of Fermentation and Bioengineering (1990) 70 (3), pp. 190-192: The preparation of xcex3-CD using the xcex2- and xcex3-CD-forming CGTase from Bacillus sp. AL-6 in the presence of ethanol, which results in an increased production of xcex3-CD.
CA 107:57466 describes the preparation of xcex3-CD using the xcex3-CGTase from Bacillus sp. 313.
EP 291,067: Preparation of xcex3-CD using the CGTase from Bacillus macerans. Product specificity for xcex3-CD is achieved by adding a complexing agent, for example cyclohexadec-8-en-1-one.
DE 40 09 822 (corresponds to U.S. Pat. No. 5,409,824): Production of xcex3-CD using the xcex3-CGTase from Bacillus sp. 290-3.
Both in comparison to xcex1-CD and in comparison to xcex2-CD, xcex3-CD possesses specific advantages which identify it as the only possible CD, or the most suitable CD, for a series of applications.
In comparison to xcex1-CD, which is made of six glucose units, xcex3-CD, which consists of eight glucose units, possesses a larger hydrophobic cavity which also makes it possible to complex guest molecules which, for steric reasons, cannot be complexed by xcex1-CD.
In comparison to xcex2-CD (solubility in water at room temperature: approx. 18.5 g/l), xcex3-CD possesses a substantially higher solubility in water (at room temperature: approx. 232.0 g/l) and is consequently more suited than xcex2-CD for complexing reactions in aqueous solutions. A further advantage of xcex3-CD, when compared with xcex2-CD and modified xcex2-CD derivatives, is its low toxicity. In an animal model, xcex1-CD derivatives and xcex2-CD derivatives are more toxic than xcex3-CD when administered either orally or intravenously.
In the Sequence Listing, Xxx refers to a deleted amino acid.
Other objects and features of the present invention will become apparent from the drawing and from the following Examples, which disclose the embodiments of the present invention. It should be understood, however, that the drawing and the Examples are designed for the purpose of illustration only and not as a definition of the limits of the invention.